In a gene expression array study comparing signatures of chronic liver diseases with hepatocellular carcinoma, we revealed a molecular signature that separates these patients for their risk of developing advanced disease. Epithelial cell adhesion molecule was identified as the lead gene in this signature and silencing of this gene resulted in growth suppression of hepatocellular carcinoma cells. Our results support the notion that large numbers of procarcinogenic genes can be acquired before a solitary tumor is formed, and suggest that an alternative mechanism exists for hepatocellular carcinoma, whereby multiple tumor-initiating events can simultaneously occur at the beginning of tumor initiation. The identification of these procarcinogenic genes in premalignant tissues and the mechanisms for their activation or inactivation may improve the future diagnosis of high-risk populations and may guide new strategies for chemoprevention. We have also used global molecular profiling to analyze the gene expression differences among EpCAM positive or negative hepatocellular carcinoma. We found that EpCAM could significantly differentiate hepatocellular carcinoma into two subtypes that resembled liver lineages. In particular, EpCAM-positive hepatocellular carcinoma displayed distinct features of hepatic stem/progenitor cells including an activation of the wnt-beta catenin pathway. Meanwhile, EpCAM-negative hepatocellular carcinoma displayed features of mature hepatocytes. We also found that hepatocellular carcinomas could be further stratified into four distinct subtypes with the additional assessment of alpha-fetoprotein status. These four subtypes were associated with prognostic outcome of hepatocellular carcinoma and cells double positive for EpCAM and alpha-fetoprotein had the worst prognosis. Furthermore, these subtypes resembled certain stages of liver lineages and EpCAM/alpha-fetoprotein-positive cells displayed a distinct molecular signature with features of hepatic stem/progenitor cells. Moreover, these cells, characterizing a poor prognostic hepatocellular carcinoma subtype, were capable of initiating highly invasive hepatocellular carcinoma in in-vitro and in-vivo models. This work suggests that EpCAM and alpha-fetoprotein are useful diagnostic markers for hepatocellular carcinoma which can be used as a convenient classification system for prognosis. Furthermore, EpCAM and alpha-fetoprotein may act as downstream molecules to maintain HCC stemness and serve as good markers for HCC initiating cells. We also explored the mechanism by which EpCAM is elevated in hepatocellular carcinoma subtypes with stem/progenitor cell features. We found that the activation of wnt-beta-catenin pathway regulates EpCAM expression. We demonstrate that EpCAM is a biosensor for wnt-beta-catenin signaling and is transcriptionally up-regulated by this pathway through direct Tcf binding element interactions. Our data suggest that the convergence of EpCAM expression and wnt-beta-catenin signaling functions to maintain hepatocellular carcinoma cell growth. Inhibition of hepatocellular carcinoma cell growth could be achieved through blockade of EpCAM/wnt-beta-catenin signaling in EpCAM-positive hepatocellular carcinoma cells. With these findings, we propose that EpCAM/wnt-beta-catenin signaling functions to maintain hepatocellular carcinoma stem cell growth and that EpCAM expression-based classification of hepatocellular carcinoma could be useful in clinical settings to stratify hepatocellular carcinoma patients who may benefit from beta-catenin/EpCAM adjuvant therapies. MicroRNAs are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. A global microRNA microarray approach was used to explore whether certain microRNAs were associated with hepatocellular carcinoma stem cells. We found that the conserved microRNA-181 family members were up-regulated in hepatoceullar carcinoma stem cells. Inhibition of microRNA-181 led to a reduction in number and tumor initiating activity of hepatocellular carcinoma stem cells while addition of microRNA-181 led to an enrichment of this cell type. In further studies, we showed that microRNA-181 could directly target transcriptional regulators of differentiation in the liver and an inhibitor of wnt-beta-catenin signaling. These results suggest a novel regulatory link between microRNA-181 family members and liver cancer stem cells and imply that molecular targeting of microRNA-181 may eradicate hepatocellular carcinoma.